Data independent acquisition (DIA) is a mass spectrometry technique in which MS/MS data is acquired for substantially all precursor ions across a mass (m/z) range of interest. An implementation of DIA is symbolically depicted in FIG. 1. Within an acquisition cycle, an isolation window is stepped across a mass range of interest in a series of MS/MS events. For each MS/MS event, ions within a mass window are isolated (i.e., mass selected), fragmented (e.g., by collisionally activated dissociation), and an MS/MS spectrum is acquired of the resultant product ions. The isolation windows are set such that they sequentially span the precursor ion mass range of interest. In the example depicted, the precursor range of interest is 500-1000 Thomson (m/z units, abbreviated Th), and 25 isolation windows of 20 Th width are used. After the isolation window has been stepped across the entire range of interest, the acquisition cycle is repeated. As depicted in FIG. 1, each acquisition cycle may be initiated by a full range MS scan (a “survey scan”) to obtain a spectrum of the precursor ions. DIA has the advantage of producing a rich data set without requiring a priori knowledge of targets, and the data set can be revisited to investigate hypotheses generated post-acquisition.
The selection of the isolation window width in DIA involves balancing competing performance considerations. In general, using narrower window widths reduces the number of different precursor ion species fragmented together in an MS/MS event, rendering the resultant product ion spectra relatively clean and easy to interpret (noting that since each MS/MS spectra will typically include ions from a number of precursor ion species, some sort of deconvolution to assign product ions in the spectra to a corresponding precursor ion species will typically be required). However, because the number of MS/MS events in a DIA cycle is inversely proportional to the window width, each DIA cycle will take a relatively long time to complete when narrow window widths are utilized. This reduces the number of DIA cycles that can be completed across a chromatographic peak, which in turn compromises the ability to reliably and accurately quantify sample components, particularly when elution peaks are narrow. Conversely, wide isolation windows permit a relatively large number of acquisition cycles to be performed across a chromatographic peak, but the resultant MS/MS spectra are highly complex and difficult to interpret due to the multiplicity of different precursor ion species present in an MS/MS scan; this complexity may be particularly problematic for samples in the form of a biological matrix such as blood plasma. The selection of an appropriate window width is made more difficult by the fact that precursor ion species produced from a sample will not typically be distributed evenly across the m/z range of interest, and information regarding their distribution may not be available prior to running the analysis.